Production of chloramphenicol, bottromycin and fradicin

ABSTRACT

The production of chloramphenicol, bottromycin and fradicin by fermentation utilizing a chloramphenicol-producing microorganism belonging to a new Kitasatoa group of the new species of the Actinoplanaceae. A live culture of the organism is on deposit with the American Type Culture Collection under the number ATCC 21405.

United States Patent Hata et al.

[451 Mar. 21, 1972 1 PRODUCTION OF CHLORAMPHENICOL, BOTTROMYCIN AND FRADICIN Inventors: Toju Hata, 94 Nakameguro, 4-chome;

Filed:

Akihiro Matsumae, l0 Isurumaki, 5- chome; Kimihiko Abe, 50 Kameido-machi, 7-chome; Yoshimoto Sano, 18-17 Nishiogi, Kita, all of Tokyo; Mariko Ohtani, 4055 Mutsuura-cho, Yokohama; Satoshi Ohmura, 207 Tamagava, Setamachi, Tokyo, all of Japan Aug. 18, 1969 Appl. No.: 851,100

US. Cl ..195/80, 195/96 Int. Cl. ..C 12d 9/00 Field of Search 195/80, 81

[56] References Cited OTHER PUBLICATIONS Abe et al., Studies on Antibiotics Produced from Kitasatoa Kauaiensis Sp. Nov., J. Antibiotics, 21(a), 1968, pp. 545- 550 Primary Examiner-Joseph M. Golian Attorney-Howard E. Post and Robert H. Dewey [57] ABSTRACT 1 Claims, No Drawings with a new organism of the new genus Kitasatoa.

Usually, for the fermentation of the antibiotics chloramphenicol, bottromycin and fradicin, the actinomycetes belonging to a genus Streptomyces in the family Strepto mycetaceae are utilized. The present inventors have now isolated a new strain of soil organism, given the laboratory designation KA-28l, and found that this organism belonged to the family Actinoplanaceae but not the Streptomycelaceae, and this organism produced chloramphenicol, bottromycin and fradicin. This invention is based on this discovery. A culture of the strain KA-28l has been deposited with the Japanese Type Culture Collection Fermentation of Research Institute, Agency of Industrial Science as FERM No. 177 on June 4, 1968. the address of this institute is:

4-5-lrage-Higashi, Chiba, Chiba-ken, Japan. A live culture of the organism is also on deposit with the American Type Culture Collection under the number ATCC 21405.

Morphological and biological studies of this new strain, KA-28 l are as follows:

1. The vegetative mycelium i The vegetative mycelium was 0.5-1.2 y. in width and well diverged. The septum of the mycelium was usually not recognized, even in the older culture too.

2. The aerial mycelium The aerial mycelium was 0.5-1.2 ,u in width and grew lineally from the vegetative mycelium. lt diverged in all directions, and the common spirals or whorls were not recognized.

3. Conidiospore The conidiospore developed on the aerial mycelium was cylindrical or ellipsoidal form. The surface of the con idiospore was smooth or rough.

4. Sporangiospore Sporangiospore formed in a single zoospore or in pairs on the aerial or the vegetative mycelium. This zoospore swam rapidly in the water. On the electron-microscopic observation, the zoospore had a single or double flagella (longer than 100 M- 5. Other of the organisms indicated various morphological states and were as follows:

1. The vesicular body (30-50 [.L) was formed by gathering mycelium. This vesicular body contained a moist substance, and was covered with a thin membrane. Later, the mycelium netlike body or sclerotic granule, which was covered with sclerotic vessel, was formed from this vesicular body.

2. On the top of the conidiosphore, sporangium-like organs which produced 5-10 spores were recognized.

3. Crystalline bodies were recognized.

Cultural Findings:

The above strain is able to grow at l0-40 C., and the most suitable growth temperature is -32 C.

A colony of this strain on a synthetic media was moist and grew poorly in the early stage, while in the later stage gradually dried to form powdery aerial mycelium, colored reddish or purplish brown. A colony on a glucose nutrient agar was large and yellowish brown, and it protruded on the center and became wrinkled from its center to circumference. White powdery aerial mycelium was formed on the surface in the early culture,.arid brownish soluble pigment was produced.

' ln nutrient broth, a pellet of mycelium was formed on the glass wall and on the bottom of the test tube.

In Table 1, culture characteristics, in Table 2, physiological characteristics, and in Table 3, utilization of carbon sources,

are listed.

TABLE 1 KA-Zfi l Conidia ellipsoidal Zoospore cylindrical single or in pairs 3.0-2.0 x 0.9 ,i G. poor. reddish brown Glycerol Czapeks A.M. white agar S.P. reddish brown G. dewdroplike. Glucose grayish red brown Czapeks agar A.M. grayish white S.P. reddish brown G. large, wet. elevated, Glucose pale yellow asparagine agar A.M. brownish gray S.P. yellowish brown G. brown Ca-Malate agar A.M. brownish white S.P. light brown G. yellow Starch agar A.M. light brownish white S.P. grayish yellow 7 G. ale brown Glucose nutrient A.M. white agar S.P. brown G. light brownish gray Peptone agar A.M. brownish white S.P. pale brown Bennett's G. light brownish gray Bennett's agar A.M. brownish white S.P. brown G. pale olive Oat meal agar A.M. white S.P. brown G. yellowish brown Potato plug A.M. white S.P. grayish yellow brown G. pale brown Carrot plug A.M. white S.P. none G. ring and sedimentation yellowish brown Milk A.M. white S.P. yellowish brown G: Growth, A.M.: Aerial mycelium, S.P.: Soluble pigment.

TABLE 2 Growth condition KA-28l temp. 1047 c.

TABLE 3 Arabinose Xylose KA-28l Glucose Mannose Fructose Lactose Saecharose lnositol Rhamnose Salicine Mannitol colony formation, production ol'aerial mycelium colony formation no growth The new Kitasatoa kauaiensis, KA-28 l is recognized as belonging to the family Actinoplanaceae, differentiated from the family Streptomycetaceae, on account of above mentioned morphological and biological characteristics and especially the nature of producing sporangium. (see Bergey's Manual of Determinative Bacteriology (1957) and Actinomycetes ll).

Of the family Actinoplanaceae producing sporangium, Acrinoplanes (J. Elishia Mitchell Sci. Soc., 66, 87 1950), Amplariella (J. Elishia Mitchell, Sci. Soc., 79, 53 (1963), Spirillospora (J. Elishia Mitchell Sci. Soc., 79, 53 (1963), and Dactinosporangium (Archiv.fl'i1' Mikrobiol. 58, 42 (1967), were reported as having motile zoospores, but the said new strain is different from these genera for the reason of producing sporangium on the vegetative mycelium.

The strains belonging to the genus planomanospora listed in the key table (Archiv. ft'ir Microbiol. 58, 42 (1967) produce a cylindrical sporangium on the aerial mycelium, and the single cylindrical zoospore is in the sporangium. On the other hand, strain KA-28l is diplococcus-like, and in a sposangium many zoospores are included. Accordingly, this new strain, KA-28 l is different from the genus planomonospora. The location of the sporangium and the morphological state of this new strain is recognized to be different from that of the known genus belonging to the family Actinoplanaceae. Then this new strain is concluded to be a new genus belonging to the family Actinoplanaceae. The shape and the size of the zoospores and the surface of the conidium are different. Then Kitasatoa gen. Nov. is newly established in the family Actinoplanaceae, and K ilasatoa kauaiensis sp. Nov. is established, and-this new strain belongs to this species. A culture of this strain of the living organism isolated from soil and given the laboratory designation KA-28l has been deposited with the Japanese Type Culture Collection, (Fermentation of Research institute, Agency of industrial Science and Technology) and added to its permanent collection ofmicro-organisms as FERM 177,

KA-28 l: Kitasatoa kauaiensis Matsumae et al.

The strain No. KA-28l is characterized as a source of chloramphenicol, bottromycin and fradicin. The term Kitasatoa kauaiensis has the meaning: the standard strain No. KA-281 as well as natural and artificial variants thereofwhich produce chloramphenicol, bottromycin and fradicin,

To produce chloramphenicol, bottromycin and fradicin according to the present invention, Kitasatoa kauaiensis is cultured in a suitable aqueous nutrient medium. As convenient media, those well known for cultivation of organisms of Kirasaroa in stationary or submerged culture are available for the production of chloramphenicol and bottromycin. The fermentation may be advantageously carried out under aerobic conditions with agitation over a period of 2 to 5 days at temperatures in the range of 25 to 35 C., although the range of 26 to 30 C. is preferable. in the preparation of chloramphenicol and bottromycin any nutrient solution in which a strain belonging to Kitasataa kauaiensir grows may be employed. As carbon sources for the fermentation medium, for example glucose, starch, dextrin, molasses, fructose, sucrose, glycerol, lactose and maltose are preferable and nitrogen sources for example meat extract, peptone, soya bean, yeast, ammonium salt, and nitrate are available. Two or more of these nitrogen sources can be used effectively, as inorganic salt; e.g., potassium salt, sodium salt, calcium salt.

Phosphate which dissociates into ions may be mixed into a culture media. Various metal ions, for example, magnesium, zinc, cobalt, iron, may also be added in minute or trace quantity to the culture medium ifdesired.

For example, aerobic fermentation of Kitasatoa kauaiensis may be effected in an aqueous medium containing 2% soya bean meal, 2% starch, 0.3% yeast, 0.3% NaCl 0.3% CaCO at 28 C. for 96 hours. Chloramphenicol and bottromycin are produced in the same culture broth. The antibacterial activity of chloramphenicol and bottromycin was estimated by a paper disc plate method using E. soli growth or nutrient agar at pH 8.0, respectively.

For the isolation of chloramphenicol from the culture broth conventional isolation and purification methods for antibiotics are used. For example, a culture broth is separated into filtrate and mycelium by centrifugation or filtration. Chloramphenicol can be extracted from the filtrate by the use of a hydrophobic polar solvent such as ethyl acetate, butyl acetate, etc. and dried in vacuo. Various methods, such as repeated recrystallization, silica gel or alumina chromatography have been used to isolate chloramphenicol from the crude powder. For the isolation of bottromycin and fradicin from the culture broth, conventional isolation or purification methods for antibiotics are applied. For example, culture broth is separated into filtrate and mycelium by centrifugation or by filtration. The filtrate is extracted by a hydrophobic solvent such as ethyl acetate, chloroform, ethylene chloride, etc. The mycelium is extracted with acetone or methanol, the solvent concentrated in vacuo, and then extracted with the same solvent as the filtrate. Then the extract is dried in vacuo. Or it an be extracted with acidic water from the solvent extract, and then the aqueous layer neutralized and extracted with a solvent such as ethyl acetate in weak alkali, and dried in vacuo to obtain a brownish crude powder containing bottromycin and fradicin.

For the isolation of bottromycin and fradicin from this crude powder, the following methods can be used in combination; that is alumina, silica gel, gel filtration or partition chromatography, etc.

Crude material is placed on an alumina chromatography column and then developed with chloroform and methanol. From the former solvent was obtained bottromycin and from the latter, fradicin. Both are further purified by Sephadex LH 20 column chromatography using methanol. The bottromycin is obtained as pale yellowish needles and recrystallized from hot ethyl ether-n-hexane, while the fradicin is obtained as yellow needles, and recrystallized from methanol-chloroform.

Chloramphenicol which was obtained by such procedures had the following characterstics:

l. Elemental analysis MeOH max

7. Infrared absorption spectrum with potassium bromide:

\/KBr max 8. Color reaction N inhydrin, Biuret, Molisch and FeCl;, tests. Negative 3200, I680, 1510, 1340, 1240, 1100. wasta e-1 9. Nature: Neutral substance. 10. Antimicrobial spectrum:

Minimum Concentration For complete Inhibition Bottromycin, which was obtained by such procedures had the following characteristics:

1. Formula C H N O S 2. Molecular weight 781 3. Elementary analysis:

Found:

Calcd:

C. 60.89 C.61.3l

max 1490, 1440, 1370, 1250, 700 cm".

10. Nature Basic substance 1 l. Antimicrobial spectrum:

Minimum Concentration For Complete Inhibition Test Organisms mcgJml.

Sarzina lulru PC1 1001 1.25 Micromt'tus fluru: 1.25 Slaplrrhmicvus aureu: 209 P 0.6 Bacillus .ruhlih's PC] 219 0.08 Errhz'rirhia mli 50 Shigella flexm'rl' 100 Vibriu ("um/nu lnaha 5 Khhsiellu pnvunmnim' 100 Salmonella lyplmru 100 M ycohai'leriuni ATC C 607 50 Fradicin which was obtained by such procedures had the following characteristics:

1. Elementary analysis: C. 71.46%, H. 5.59%, N. 11.67%

2. Molecular weight: 500 (Berger method) 3. Melting point: 210 C. (dec) 4. Optical rotation: [01] =+65 C., (C=1 dioxan) 5. Solubility: Easily soluble in water, methanol, ethanol,

soluble in chloroform, ethylene chloride.

6. Color reaction: Positive. Zeisel reaction.

7. Nature: Weak basic substance.

8. Ultraviolet absorption spectrum in methanol:

270 (sh), 295 1720), 325 (sh). 337 3 10), 3 56 (179 37s 1111...

Infrared absorption spectrum with potassium bromide:

1450,1310,1280,1l10, max 1020, 840, 740 emf.

10. Antimicrobial spectrum:

Minimum Concentration For Complete Inhibition Test Organisms (meg/ml.)

Aspergillur aryzae 5 Aspergillus niger l .25 Penicillium glaucum 0.5 Saccharomycer sahe l0 Willia anamalu l0 ToruIa rubra 2.5 Mucnr nucedo l0 Trichophymn inlerdigilale 2.5 Staphylococcus aureus Escherichia call 100 This substance was identified as fradicin from the properties described above. (Science 1 13, 261, 1951).

EXAMPLE 1 An aqueous medium containing 2% glucose, 0.5% peptone, 0.5% meat extract, 0.5% NaCl, 0.3% CaCO at pH 7.0 is divided into 500 m1. shake flasks in portions of ml. per each, sterilized for 15 min. at 120 C., cooled down to 28 C., inoculated with colonies of Kitasatoa kauaiensis (K28l) (FERM 177) maintained on an agar slant (Krainskys medium) and shake cultured at 27 C. for 72 hours to obtain a seed culture. Twenty liters of medium with the above-described composition is put in a 30 liter jar fermentor, sterilized at 120 C. for 15 minutes, then cooled to 28 C. and 250 ml. of abovedescribed inoculum is added.

After 48 hour fermentation at 2830 C. aerating (20 liter per minute aeration), and agitating 230 r.p.m. to obtain a seed culture, two hundred liters of medium which consists of 2% starch, 2% soybean meal, 0.3% yeast, 0.5% NaCl, 0.3% CaCO is put in a 400 liter stainless steel fermentor sterilized at 120 C. for 15 minutes, then cooled to 28 C. and 20 liters of the above seed is inoculated. After 72 hours of fermentation, at 27-30 C. aerating (200 liters per minute) and agitating 200 r.p.m., mycelium is removed to obtain 360 liters of culture filtrate (bottromycin activity 7.2 mcg./ml.).

The culture filtrate is extracted twice with ethyl acetate at pH 7.2, 80 1. and 40 1. respectively. Ethyl acetate extract is concentrated to 17 liters under vacuum (about US volume). This solution is then extracted with 0.1 mole acetic acid (1/5 volume) four times.

The acetic acid solution (14.5 liters) is adjusted to pH 7.8 with 3 N X NaOH. The solution is then extracted with ethyl acetate (1/5 volume) three times. Crude brownish bottromycin (5.9 g) is obtained after the solution is concentrated.

The crude bottromycin is dissolved with chloroform and put on alumina column chromatography (alumina 120 g. column 3 X 15 cm.). An active fraction (bottromycin 1.8 g.) is obtained. The above mentioned powder is dissolved with methanol and put on a Sephadex LH 20 column chromatography. Yield 62 percent. Bottromycin (1.5 g.) is obtained as a colorless powder.

EXAMPLE 2 20 L. of aqueous medium containing 2% starch, 2% soya bean, 0.3% yeast, 0.5% NaCl, 0.3% CaCO at pH 7.0 in 30 liter jar fermentor was sterilized at 1 20 C. for 15 minutes.

KA-281 strain was cultured under the same condition as Example 1, and the cultural process was as follows:

From this culture filtrate, bottromycin was obtained as in Example 1.

EXAMPLE 3 20 L. of aqueous medium containing 2% starch, 2% corn steep liquor, 0.3% yeast, 0.5% NaCl, and 0.3% CaCO at pH 7.0 in a 30 liter jar fermentor was sterilized at 120 C. for 15 minutes.

KA-281 strain was cultured under the same condition as Example 1 and the cultural process was as follows:

Bottromycin Fermentation Time Starch Remaining (hours) pH (mg/ml.) (meg/ml.)

From this culture filtrate, bottromycin was obtained as in Example 1.

EXAMPLE 4 1.75 Liters of cultured broth obtained from the same fermentation process as Example 1 was extracted with 6 l. and 4 l. of ethyl acetate at pH 8. Seven liters of the extract was dried in vacuo and 25 g. of crude crystals of chloramphenicol was obtained. These crude crystals were divided into two parts and one was repeatedly recrystallized with an acetone-chloroform system. 370 mg. of colorless needles was obtained. The yield was 24 percent.

The other part was dissolved in a little acetone and put on silica gel chromatography column. The column (2.5 X20 cm.) was prepared 50 g. ofsilica gel with benzene. It was developed with acetone; benzene I22) and the active fraction was concentrated. After standing with chloroform 950 mg. of brownish needles was obtained and recrystallized with acetone-chloroform and 650 mg. of colorless needles was obtained. Yield was 42 percent.

EXAMPLE 5 Twenty liters of medium prepared with 2% starch, 2% soya bean meal, 0.3% yeast, 0.5% sodium chloride, and 0.3% calcium carbonate was poured into 30 l. jar fermentor, and adjusted to pH 7.0 and sterilized for 15 minutes at 120 C. Then KA-28l strain was cultured as the same condition as Example 4. The result was as follows:

From this fermentation filtrate, chloramphenicol was obtained with the same procedure as Example 4.

EXAMPLE 6 Three hundred and sixty liters of the filtrate obtained from the same fermentation process as Example 1 was extracted with 801. and 401. portions of ethyl acetate at pH 7.2.

The extract liters) was concentrated to 17 liters and extracted four times with 0.1 M acetic acid solution. The acetic acid phase (M5 1.) was adjusted to pH 7.8 with 3 N sodium hydroxide and again extracted three times with N5 volume of ethyl acetate. The crude powder was dissolved in chloroform and chromatographed on alumina column (alumina g. column 3 X 15 cm.) and developed with methanol. 1.8 G. of fradicin was obtained. Then the powder obtained was dissolved in methanol and chromatographed on Sephadex LH 20 using methanol. mg. of fradicin was obtained as a pale yellow powder. It was dissolved in chloroform and methanol added. 50 mg. of pale yellow needles was obtained. Yield was 9 percent.

What we claim is:

1. The method of manufacturing chloramphenicol, bottromycin and fradicin by fermentation which comprises culturing the micro-organism Kitasatoa kauaiensis ATCC 21405 in a fermentation medium.

UNITED STATES PA'IENT OFFICE CERTIFICATE OF CORRECTION Patent No- 3 59 994 mm iaarch 21. 1372 Invent To'iu 'Hata et al It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 4, line 7 "NaCl" should be NaCl,- Column 4, line 12 "soli" should be coli- Column 4, line 31 "an" should be can Column 4, line 53 "40.94%" should be -40.95%-- Column 4, line 55 "232" should be -323 Column 5, line 9 "290 should be -209 Column 6, line 38 "(K-281)" should be (KA28l)- Column 7, line 41, last column in the table in Example 3,

"1.0" should be l,9 Column 8, line 18 in the table Column 2, the heading has been omitted, should be "*PH Column 3, the heading should be Starch Remaining g/ Column 4, the heading should be Chloramphenicol (mcg/ml) Signed and sealed this 1st day of August 1972.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. ROBERT GOTTSCHALK Attesting Officer Commissioner of Patents 

